Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is concerned with development of vaccines against HPV and other targets and elucidation of the PV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, referred to hereafter as pseudovirions. This methodology represents a technical breakthrough in papillomavirus research, and we have exploited this development in several ways, as outlined below. Pseudovirus production technology is being used to explore the basic features of papillomavirus virion assembly. We have determined that encapsidation of the double stranded circular genome is size-dependent, but surprisingly sequence-independent. Plasmids with no papillomavirus sequences are efficiently packaged, making these pseudovirions exceptionally flexible gene transfer vehicles. We have also determined that the capsids mature through an ordered sequence of disulfide bond formation that results in stabilization of the capsids and makes them resistant to proteolysis. In other basic virologic studies, we examined the early events in papillomavirus infection. Taking advantage of the pseudovirus approach, we determined that papillomaviruses uncoat in early endosomes and that the viral genome is subsequently directed by L2 to subnuclear domains called ND10 bodies or PODs. We further determined that co-localization of the viral genome at ND10s is required for efficient transcription of the viral genome after infection by authentic BPV1 virions. Other DNA viruses interact with ND10s early after infections, but these interactions, which do not involve capsid proteins, lead to disruption of the structures. Therefore, it has generally been found that ND10s have an antagonist function early in viral infections. Our findings are the first clear evidence that, in some instances, ND10s can promote establishment of viral infection. We have also used the pseudoviruses to screen for inhibitors of infection, which showed that an inhibitor of furin (a cell-encoded proprotein convertase) was a potent antagonist. This observation led us to determine that papillomavirus infection requires furin. This requirement is attributable to furin-dependent cleavage of a previously unrecognized consensus furin cleavage site located in the N-terminus of the L2 minor capsid protein of all papillomaviruses. The requirement for furin is the first example of a requirement of a sequence-specific cleavage of a virion protein for entry of any virus. However, this function may be an example of convergent evolution, as many bacterial toxins require furin for their escape from the endosome, and furin inhibition prevents the uncoated papillomavirus particle from escaping the endosome. We have developed a new papillomavirus neutralization assay based on the pseudovirus technology. This is the first high throughput papillomavirus neturalization assay and the first assay that is not severely limited by the availability of infectious capsids. It is sensitive, type-specific, and highly reproducible in a 96-well microtiter plate format. We expect this assay to greatly facilitate immune response monitoring and assessment of immune correlates of protection in the phase III VLP vaccine trials currently being conducted by the NCI, Merck, and GlaxoSmithKline. The assay has also been used to show that the N-terminus of the minor structural viral protein, L2, can induce neutralizing antibodies against a broad spectrum of papillomaviruses.
Showing the most recent 10 out of 37 publications
